The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.